Crystalline inhibitor of 4-hydroxyphenylpyruvate dioxygenase

ABSTRACT

The present invention relates to an improved synthesis and crystallization process of the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione, also known as nitisinone or NTBC.

FIELD OF THE INVENTION

The present invention relates to an improved synthesis andcrystallization process of 4-hydroxyphenylpyruvate dioxygenase (HPPD)inhibitor 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione,also known as nitisinone or NTBC. This new process of synthesis andcrystallization gives rise to an extremely pure and stable2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione.

BACKGROUND OF THE INVENTION

NTBC is a drug marketed by Swedish Orphan Biovitrum International ABunder the brand name Orfadin® and it is used to slow the effects ofhereditary tyrosinemia type 1 (HT-1) in adult and pediatric patients. Ithas been approved by FDA and EMA in January 2002 and February 2005respectively.

HT-1 disease is due to a deficiency of the final enzyme of the tyrosinecatabolic pathway fumarylacetoacetate hydrolase. NTBC is a competitiveinhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD), an enzyme whichprecedes film arylacetoacetate hydrolase. By inhibiting the normalcatabolism of tyrosine in patients with HT-1, NTBC prevents theaccumulation of the toxic intermediates maleylacetoacetate andfumarylacetoacetate, that in patients with HT-1 are converted to thetoxic as succinylacetone and succinylacetoacetate, the former inhibitingthe porphyrin synthesis pathway leading to the accumulation of5-aminolevulinate.

Usefulness of NTBC in the treatment of further diseases has also beendocumented. A non-comprehensive list is reported hereinafter.

Effectiveness of Orfadin® in the treatment of diseases where theproducts of the action of HPPD are involved (e.g., HT-1) has beendescribed notably in EP0591275131 corresponding to U.S. Pat. No.5,550,165B1. Synthesis of NTBC is also described in this patent.

WO2011106655 reports a method for increasing tyrosine plasmaconcentrations in a subject suffering from oculocutaneous/ocularalbinism, the method comprising administering to the subject apharmaceutically acceptable composition comprising NTBC in the range ofbetween about 0.1 mg/kg/day to about 10 mg/kg/day.

U.S. Pat. No. 8,354,451B2 reports new methods of combating microbialinfections due to fungi or bacteria by means of administration to asubject of a therapeutically active amount of NTBC.

WO2010054273 discloses NTBC-containing compositions and methods for thetreatment and/or prevention of restless leg syndrome (RLS).

EP1853241B1 claims the use of NTBC in the treatment of aneurodegenerative disease, notably Parkinson disease.

Introne W. J., et al., disclosed usefulness of nitisinone in thetreatment of alkaptonuria (Introne W. J., et al., Molec. Genet. Metab.,2011, 103, 4, 307). The key step of the synthesis reported inEP0591275B1 (now propriety of Swedish Orphan Biovitrum International AB,SE), involves the reaction of 2-nitro-4-trifluoromethylbenzoyl chlorideand cyclohexane-1,3-dione in the presence of triethylamine and then useof acetone cyanohydrin in order to promote the rearrangement of the keyintermediate enol ester. After washing and extraction from CH₂Cl₂, thecrude product is recrystallized from ethyl acetate to get the desired2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione as a solidhaving a melting point of 88-94° C.

Another patent (U.S. Pat. No. 4,695,673) filed in name of StaufferChemical Company disclosed a process of synthesis of acylated1,3-dicarbonyl compounds in which the intermediate enol ester isisolated prior to its rearrangement into the final product, saidrearrangement making use of a cyanohydrin compound derived from alkalimetal, methyl alkyl ketone, benzaldehyde, cyclohexanone, C₂-C₅ aliphaticaldehyde, lower alkyl 341 or directly by using hydrogen cyanide.

Yet another patent (U.S. Pat. No. 5,006,158) filed in name of ICIAmericas Inc. disclosed a process similar to the one disclosed in U.S.Pat. No. 4,695,673 wherein the intermediate enol ester was isolatedprior to its rearrangement into the final product by use of potassiumcyanide. Said reaction can optionally be done by concomitant use of aphase transfer catalyst such as Crown ethers. The preferred solvent forconducting such a reaction is 1,2-dichloroethane.

Still a further patent (EP0803791) filed in name of Zeneca Ltd disclosedan alternative synthesis of nitisinone involving the reaction of1,3-cyclohexanedione and variously substituted benzoyl chloride in thepresence of sodium or potassium carbonate in CH₃CN or DMF. Best yieldswere obtained using CH₃CN as solvent and sodium carbonate as the base.Reaction was performed at 55-57° C. in 17 hours.

It is well known that one of the problems of the actual drug formulation(i.e., Orfadin® capsules) is its chemical instability. Indeed, even ifOrfadin® has to be stored in a refrigerator at a temperature rangingfrom 2° C. to 8° C., its shelf life is of only 18 months. After firstopening, the in-use stability is a single period of 2 months at atemperature not above 25° C., after which it must be discarded. It willbe evident that such storage conditions have an impact in thedistribution chain of the medicine, in terms of costs and also in termsof logistics for the patient. Therefore, there is an urgent need of morestable formulations, both from a logistic supply chain point of view,and from the patient compliance point of view. Since the formulation ofOrfadin® contains only the active ingredient and starch as excipient,relative instability may be attributed to the active pharmaceuticalingredient itself; in other words it can derive from the way it issynthesized and/or the way it is extracted from the reaction mixture,and/or the way it is finally crystallized. Furthermore, some impuritiesmay contribute to render the final product less stable overtime.Consequently, it is of major importance to identify a process ofsynthesis and/or a crystallization method that enable the reliableproduction of a highly pure and stable product.

Impurities as herein-above mentioned can derive either from the finalproduct itself (through chemical degradation) or directly from thestarting materials/solvents used in the process of synthesis. Regardingthe latter option, it is therefore primordial to ascertain that at eachstep, impurities are completely removed in order not to get them at thefinal stage, also considering that some of them could potentially becyto/genotoxic.

The impurities correlated to nitisinone can be either derived from thestarting materials themselves (i.e., impurities 1 and 2) or obtained asside products during the process of synthesis major under storageconditions (i.e., impurities 3 to 5) and are the following:

-   2-nitro-4-(trifluoromethyl) benzoic acid (Impurity n° 1),-   1,3-cyclohexanedione (CHD) (Impurity n° 2),-   4-(trifluoromethyl)salicylic acid (Impurity n° 3),-   2-[3-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione    (Impurity n° 4), and-   6-trifluoromethyl-3,4-dihydro-2H-xanthene-1,9-dione (Impurity n° 5).

Impurity-2, impurity-3, and impurity-5 have been previously reported inWO2015101794. Strangely, impurity-4 has never been reported, even if itis an obvious side-product which can easily be formed during thecoupling reaction between 1,3-cyclohexanedione and2-nitro-4-(trifluoromethyl) benzoic acid, the latter being not 100% purebut Wining some amount of regioisomer 3-nitro-4-(trifluoromethyl)benzoic acid.

Potential genotoxicity of impurity n° 4 which possesses an aromaticnitro moiety was assessed using in-silica techniques and resulted to bea potential genotoxic impurity. According to the FDA ICH M7 guidelines,daily intake of a mutagenic impurity (Threshold of ToxicologicalConcern, TTC) in an amount not greater than 1.5 μg per person isconsidered to be associated with a negligible risk to develop cancerover a lifetime of exposure. Consequently, assuming a daily dose of 2mg/kg, for a person weighing 70 kg, the maximum tolerated impuritycontent of such a compound would be of about 11 ppm, as calculatedaccording to the equation underneath.

${{concentration}\mspace{14mu}{limit}\mspace{14mu}({ppm})} = \frac{{TTC}\mspace{11mu}\left( {µ\; g\text{/}{day}} \right)}{{Dose}\mspace{11mu}\left( {g\text{/}{day}} \right)}$

It is therefore of paramount importance to ensure that the process ofsynthesis of nitisinone and the purification steps of the same give riseto an API devoid of such impurity n° 4, or at least far below thethreshold of 11 ppm as indicated above. The skilled person willunderstand that total absence of said impurity is highly desirable.

It is well known in the pharmaceutical field that investigation ofpotential polymorphism of a solid API is of crucial importance and isalso recommended by major regulatory authorities such as FDA.

Notwithstanding the fact that nitisinone has been used for years totreat HT-1 patients, it appears that no NTBC formulation fully satisfiesthe requisites of stability and/or compliance standard for the patients.Therefore, there is an unmet medical need of long-term pure and stableformulations.

DESCRIPTION OF THE INVENTION

It has been surprisingly found that nitisinone obtained through theprocess, object of a first embodiment of the instant invention is notonly highly pure, but also extremely stable. This process of synthesisencompasses a crystallization step of2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione by means oftoluene and traces amount of acetonitrile.

While not intending to be bound in any way by any theory, it is believedthat the improved chemical stability of nitisinone is attributable toits crystalline purity obtainable through the presently claimedinvention.

The expressions “extremely stable” and/or “long term stable” and/or“highly stable” shall be understood for purposes of the presentinvention to mean that the chemical integrity of nitisinone is of atleast 95% after one month of storage of nitisinone at 25° C. and 60% RH.

When referring to purity determined by HPLC techniques, saidchromatograms are gathered at a wavelength of 235 nm.

When referring to temperatures and/or pH, the term “about” shall beunderstood for purposes of the present invention to mean plus or minus10% of the temperature and/or pH values mentioned, preferably plus rminus 5% and even more preferably plus or minus 1%.

In one embodiment, said chemical integrity is of at least 96% after sixmouths of storage of nitisinone at 25° C. and 60% RH.

In another embodiment, said chemical integrity is of at least 97% aftersix months of storage of nitisinone at 25° C. and 60% RH.

In a preferred embodiment, said chemical integrity is of at least 98%after six months of storage of nitisinone at 25° C. and 60% RH.

In a still preferred embodiment, said chemical integrity is of at least99% after six months of storage of nitisinone at 25° C. and 60% RH.

Another embodiment of the invention contemplates chemical integrity ofnitisinone after nine months of storage at 25° C. and 60% RH.

In said embodiment, the chemical integrity of nitisinone is of at least95% after nine months of storage of nitisinone at 25° C. and 60% RH.

In a preferred embodiment, the chemical integrity of nitisinone is of atleast 96% after nine months of storage of nitisinone at 25° C. and 60%RH.

In another preferred embodiment, the chemical integrity of nitisinone isof at least 97% after nine months of storage of nitisinone at 25° C. and60% RH.

In a still preferred embodiment, the chemical integrity of nitisinone isof at feast 98% after nine months of storage of nitisinone at 25° C. and60% RH.

In a still more preferred embodiment, the chemical integrity ofnitisinone is of at least 99% after nine months of storage of nitisinoneat 25° C. and 60% RH.

Another embodiment of the invention contemplates chemical integrity ofnitisinone after six months of storage at 40° C. and 75% RH.

In said embodiment, the chemical integrity of nitisinone is of at least95% after six months of storage of nitisinone at 40° C. and 75% RH.

In a preferred embodiment, the chemical integrity of nitisinone is of atleast 96% after six months of storage of nitisinone at 40° C. and 75%RH.

In another preferred embodiment, the chemical integrity of nitisinone isof at least 97% after six months of storage of nitisinone at 40° C. and75% RH.

In a still preferred embodiment, the chemical integrity of nitisinone isof at least 98% after six months of storage of nitisinone at 40° C. and75% RH.

In a still more preferred embodiment, the chemical integrity ofnitisinone is of at least 99% after six months of storage of nitisinoneat 40° C. and 75% RH.

In an even more preferred embodiment, nitisinone is in crystalline formand contains less than 10 ppm of2-[3-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione and lessthan 0.05% of any single impurities chosen from the group consisting of2-nitro-4-(trifluoromethyl) benzoic acid, 1,3-cyclohexanedione,4-(trifluoromethyl)salicylic acid, and6-trifluoromethyl-3,4-dihydro-2H-xanthene-1,9-dione, after storage forsix months at a temperature of 40° C. and 75% relative humidity.

It is submitted that testing at 40° C. and 75% RH for a short time suchas six months, is considered indicative of stability at 25° C. (i.e.,room temperature) for a longer period of time (fifteen to eighteenmonths).

An embodiment of this invention consists of a process to synthesizenitisinone, by reacting 2-nitro-4-(trifluoromethyl) benzoyl chloride and1,3-cyclohexanedione in acetonitrile solution in the presence ofpotassium carbonate.

In a more preferred embodiment, nitisinone is obtained highly pure bysubsequent crystallization.

In a still more preferred embodiment, the crystallization is obtained bymeans of toluene.

In an even more preferred embodiment, the crystallization processencompasses the following steps:

-   -   a) adding crude nitisinone to an approximately 3/1 binary        acetonitrile/toluene mixture, wherein the ratio        nitisinone/binary mixture is around 1/4 (w/v) and heating at a        temperature of about 55° C. until complete dissolution;    -   b) concentrating the solution from step a) to a final volume        roughly twice the initial volume of toluene added in step a) at        a temperature below 50° C. in order to obtain a solution of        nitisinone in toluene containing approximatively 0.5-0.6 g of        nitisinone per ml of solvent;    -   c) adding toluene to the mixture obtained in step b) in order to        double the final volume obtained from step b);    -   d) repeating step b);    -   e) heating to about 55° C. for 1 h;    -   f) cooling slowly to about 10° C. in 10 to 12 h;    -   g) filtering off the solid thus obtained in step f) and rinsing        it with pre-cooled toluene; and    -   h) drying the crystals under vacuum at a temperature of about        60° C. for 4 h.

In another embodiment, the present invention provides nitisinone Form A,having an impurity 2-nitro-4-(trifluoromethyl) benzoic acid (Impurityn° 1) in an amount less than about 0.10 area percent and more preferablyless than 0.05%, as measured by HPLC/MS.

In yet another embodiment, the present invention provides nitisinoneForm A, having an impurity 1,3-cyclohexanedione (CHD) (Impurity n° 2) inan amount less than about 0.10 area percent and more preferably lessthan 0.05%, as measured by HPLC/MS.

In a still further embodiment, the present invention provides nitisinoneForm A, having an impurity 4-(trifluoromethyl)salicylic acid (Impurityn° 3) in an amount less than about 0.10 area percent and more preferablyless than 0.05%, as measured by HPLC/MS.

In a yet still further embodiment, the present invention providesnitisinone Form A, having an impurity2-[3-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (Impurity n°4) in an amount less than about 10 ppm and more preferably less than 5ppm, as measured by HPLC/MS.

In another embodiment, the present invention provides nitisinone Form A,having an impurity 6-trifluoromethyl-3,4-dihydro-2H-xanthene-1,9-dione(Impurity n° 5) in an amount less than about 0.10 area percent and morepreferably less than 0.05%, as ineasured by HPLC/MS.

In another preferred embodiment, the present invention providesnitisinone Form A, having a total amount of impurities 1 to 5 less thanabout 0.50 area percent and more preferably less than 0.25%, as measuredby HPLC/MS.

In a still even more preferred embodiment, the present inventioncontemplates nitisinone crystalline Form A having a purity of at least99.94%.

In a still further even more preferred embodiment, the present inventioncontemplates nitisinone crystalline Form A having a purity of at least99.94% and containing less than 10 ppm of impurity n° 4, preferably lessthan 5 ppm, ideally less than 1 ppm.

In a further preferred embodiment, nitisinone crystals have a PSD (d90)between 310 to 350 μm.

Another embodiment of the present invention consists of a crystallinepure nitisinone Form A, which has a X-ray powder diffraction patternwith at least five specific peaks at about 2-theta=7.4, 14.7, 15.7,22.9, and 29.7, wherein said values may be plus or minus 0.2° 2-thetaand have an intensity of at least 30%.

In a further embodiment of the present invention, the crystalline purenitisinone Form A has a X-ray powder diffraction pattern with at leastten specific peaks at about 2-theta=7.4, 14.7, 15.7, 22.9, 23.5, 23.8,25.7, 29.7, 30.3, and 31.9 wherein said values may be plus or minus 0.2°2-theta.

Another aspect of the instant invention regards usefulness of the thusobtained crystalline Form A nitisinone active ingredient in apharmaceutical formulation as a medicament due to its4-hydroxyphenylpyruvate dioxygenase inhibiting properties for thetreatment of disorders where such inhibition results in improving thehealth of the patient. In particular, patients suffering from HT-1, orfrom oculocutaneous/ocular albinism, or from microbial infections due tofungi or bacteria, or from restless leg syndrome, or fromneurodegenerative disease, notably Parkinson disease can be treated.

In a preferred embodiment of the present invention, the pharmaceuticalformulation is for treating patients suffering from HT-1.

In accordance with the foregoing, there are provided methods ofinhibiting 4-hydroxyphenylpyruvate dioxygenase enzyme in a patient. Themethods include administering an effective amount of the crystalline2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione describedherein to a patient in need thereof. Within this aspect of theinvention, patients requiring inhibition of the 4-hydroxyphenylpyruvatedioxygenase enzyme will be those suffering from diseases such asoculocutaneous/ocular albinism, microbial infections, restless legsyndrome, alkaptonuria, and hereditary tyrosinemia type 1. Preferredaspects of this embodiment include treating hereditary tyrosinemia type1.

Generally, the pharmaceutical formulation of the present invention isadministered in a “therapeutically effective amount”. The amount of thepharmaceutical formulation actually administered will typically bedetermined by a physician, in the light of the relevant circumstances,including the condition to be treated, any other potential drug thepatient is currently taking, the age, the sex, body weight, and responseof the individual patient, the severity of the patient's symptoms, andthe Like. Generally, however, the crystalline Form A nitisinone isadministered in amounts ranging from between about 0.1 mg/kg/day toabout 2 mg/kg/day, with amounts of from about 1-2 mg/kg/day beingpreferred.

A further embodiment of the present invention consists of apharmaceutical formulation comprising nitisinone obtained by the processdescribed in example 2. It is submitted that testing at 40° C. and 75%RH for a short time such as six months, is considered indicative ofstability at 25° C. room temperature) for a longer period of time(fifteen to eighteen months).

DESCRIPTION OF THE DRAWING

FIG. 1: represents the X-ray spectra of2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione Form Apolymorph.

EXAMPLES

Abbreviations:

CH₃CN: acetonitrile

HCl: hydrochloric acid

HPLC: high-performance liquid chromatography

HPLC/MS: high-performance liquid chromatography-mass spectrometry

General Remarks:

Crystalline form has been characterised by Broker D8 Advance X-raypowder diffraction (XRPD). Bragg-Brentano geometry, CuKα radiation withwavelength λ=1.54; scanning with 2θ angle range of 3° to 40°, step sizeof 0.02° for 0.5 seconds per step. Linear solid-state detector (LynxEye). Micronization has been performed with a laboratory scalemicronizer FLUID JET MILL J-20 (Tecnologia Meccanica Srl) using thefollowing milling conditions:

-   -   Grind air: dry nitrogen gas    -   Ring pressure: 3.9 bar    -   Venturi pressure: 4.1 bar    -   Feed rate: 0.30 g/min

Particle size and d(90) has been determined using laser light scatteringtechnique using a Malvern Mastersizer 3000 and water as dispersant.

Example 1

Thionyl chloride (162 g, 1.36 mol) was added dropwise into a suspensionof 2-nitro-4-trifluoromethylbenzoic acid (228 g, 0.97 mol) in toluene(630 g) at 80° C. The thus obtained solution was kept under stirring at80° C. for 20 hours, and then cooled to 50° C. The volatiles wereremoved under reduced pressure in order to get the expected2-nitro-4-trifluoromethylbenzoyl chloride as an oil. The latter, cooledto 25° C. was added dropwise to a suspension of 1,3-cyclohexanedione(109 g, 0.97 mol) and potassium carbonate (323 g, 2.33 mol) in CH₃CN(607 g). After 18 h the mixture was diluted with water (500 ml) andslowly acidified to about pH=1 with HCl 37%. The mixture was then warmedto about 55° C. and the phases were separated. The organic layer waswashed with a 10% aqueous solution of sodium chloride and then,concentrated under reduced pressure at a temperature below 55° C. toreach a volume of 380 ml. The thus obtained mixture was stirred at 55°C. for 1 h and then cooled to 0° C. in 16 to 18 h. The resulting solidwas filtered and rinsed several times with pre-cooled (0° C.) toluene.The wet solid was dried at 60° C. under vacuum for 6 h to providenitisinone. (164 g) as a white to yellowish solid with a purity of 98.4%as measured by HPLC and a content of potentially genotoxic impurity n° 4of 6.1 ppm measured by HPLC/MS.

Example 2

Nitisinone as obtained from example 1 (164 g) was added to a 3/1 (w/w)mixture of CH₃CN/toluene (volume of solvent: 038 ml). The mixture waswarmed gently to about 55° C. under stirring until solids werecompletely dissolved. The solution was then concentrated under reducedpressure maintaining the internal temperature below 50° C. to reach avolume of 290 ml. Then, more toluene (255 g) was added and the solutionwas concentrated again under reduced pressure until the residual volumereached 290 ml. The solution was heated to about 55° C. for 1 h andsuccessively cooled slowly in 10 to 12 h to 10° C. The resulting solidwas filtered and rinsed several times with pre-cooled (0° C.) toluene.The wet solid was dried at about 60° C. under vacuum for 4 h to providenitisinone (136 g) as a white to yellowish solid, with a purity of99.94% and a 99.8% assay measured by HPLC and a d(90) particle sizebetween 310 and 350 μm. The content of potential genotoxic impurity n° 4resulted below 1 ppm.

Stability Studies

As evidenced in Table 1, nitisinone obtained through the process of theinvention resulted extremely stable even in accelerated conditions for aperiod of at least six months. Importantly, the potentially genotoxicimpurity-4 resulted below the limit of quantification, independentlyfrom the storage conditions. The presence of impurity-4 was checked byreverse HPLC/MS using the method described in the table underneath.

Column Ascentis Express C18 5 μm, 50 × 4.6 mm Flow 1 ml/minute Injectionvolume 10 μl Wavelength 235 nm Column temperature 30° C. Detector MSPolarity: positive; SIM Mode; m/z = 330 Gas temp: 300° C. Gas flow: 13.1l/min Nebulizer: 20 psi Capillary: 4500 nA Step 0 to SIM > to MS Step 1to SIM > to waste Mobile phase A CH₃CN Mobile phase B H₂O/0.1% TFA HPLCGradient Time % A % B 0 50 50 10 70 30 12 70 30 13 50 50 23 50 50Retention time 5.8 minutes

TABLE 1 25° C./60% RH 30° C./65% RH 40° C./75% RH Tests Specifications 6months 9 months 6 months 9 months 6 months Appearance White to yellowishC C C C C (Visual) crystalline powder Water content (KF) NMT 0.5% w/w0.0% 0.0% 0.2% 0.0% 0.2% Assay - on anhydrous 98.0-102.0% 98.8% 98.8%99.4% 101.4% 100.4% basis (HPLC) Impurity-1 NMT 0.15% <0.01% <0.01%<0.01% <0.01% <0.01% Impurity-2 NMT 0.15% <0.01% <0.01% <0.01% <0.01%<0.01% Impurity-3 NMT 0.15% <0.01% <0.01% <0.01% <0.01% <0.01% Impurity4 NMT 10 ppm <5 ppm <5 ppm <5 ppm <5 ppm <5 ppm Impurity 5 NMT 0.15%0.01% <0.01% 0.01% <0.01% 0.01% Any unspecified NMT 0.10% <0.01% <0.01%<0.01% <0.01% <0.01% impurities Total impurities NMT 0.50% 0.01% <0.01%0.01% <0.01% 0.01% C = Conform; NMT = No more than

The invention claimed is:
 1. A method of inhibiting4-hydroxyphenylpyruvate dioxygenase enzyme in a patient, the methodcomprising administering to a patient in need thereof a therapeuticallyeffective amount of crystalline2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione having a X-raypowder diffraction pattern with at least five specific peaks at about2-theta=7.4, 14.7, 15.7, 22.9, and 29.7, wherein said peaks may be plusor minus 0.2° 2-theta and have an intensity of at least 30%.
 2. Themethod of claim 1, wherein the patient is suffering from a diseaseselected from the group consisting of oculocutaneous/ocular albinism,microbial infections, restless leg syndrome, alkaptonuria, andhereditary tyrosinemia type
 1. 3. The method of claim 2, wherein thedisease to be treated is hereditary tyrosinemia type
 1. 4. The method ofclaim 1, wherein the therapeutically effective amount of crystalline2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione is from about0.1 to about 2 mg/kg/day.
 5. The method of claim 4, wherein thetherapeutically effective amount of crystalline2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione is from about1 to about 2 mg/kg/day.
 6. A method of treating oculocutaneous/ocularalbinism, microbial infections, restless leg syndrome, alkaptonuria, orhereditary tyrosinemia type 1, the method comprising administering to apatient in need thereof a therapeutically effective amount ofcrystalline 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedionehaving X-ray powder diffraction pattern with at least five specificpeaks at about 2-theta=7.4, 14.7, 15.7, 22.9, and 29.7, wherein saidvalues may be plus or minus 0.2° 2-theta and have an intensity of atleast 30%.
 7. The method of claim 6, wherein the disease to be treatedis hereditary tyrosinemia type
 1. 8. The method of claim 6, wherein thetherapeutically effective amount of crystalline2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione is from about0.1 to about 2 mg/kg/day.
 9. The method of claim 8, wherein thetherapeutically effective amount of crystalline2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione is from about1 to about 2 mg/kg/day.
 10. A method of treating oculocutaneous/ocularalbinism, microbial infections, restless leg syndrome, alkaptonuria, orhereditary tyrosinemia type 1, the method comprising administering to apatient in need thereof a therapeutically effective amount of apharmaceutical formulation comprising crystalline2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione having X-raypowder diffraction pattern with at least five specific peaks at about2-theta=7.4, 14.7, 15.7, 22.9, and 29.7, wherein said values may be plusor minus 0.2° 2-theta and have an intensity of at least 30%.
 11. Themethod of claim 10, wherein the disease to be treated is hereditarytyrosinemia type
 1. 12. The method of claim 10, wherein thetherapeutically effective amount of crystalline2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione is from about0.1 to about 2 mg/kg/day.
 13. The method of claim 12, wherein thetherapeutically effective amount of crystalline2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione is from about1 to about 2 mg/kg/day.